流式细胞技术荧光抗体课件

流式细胞技术荧光抗体课件FluorochromesFluorochromes流式细胞技术荧光抗体课件2Fluorochrome PropertiesDesirable properties for fluorochromes: High relative brightness Narrow emission spectrum (low spectral overlap in combination) Easily conjugated (for immunophenotyping)Fluorochromes can be characterized by: Type of molecule Excitation and emission wavelengths Relative brightnessFluorochromes流式细胞技术荧光抗体课件3Fluorochrome Molecule Types Small organic moleculesexamples: FITC, BD HorizonTM V450, Cy7 Fluorescent proteinsexamples: PE, APC, PerCP Tandem dyestypically, the coupling of a fluorescent protein donor with a small organic molecule acceptorexamples: PE-Cy7, PerCP-CyTM5.5 Nanocrystals (Qdots)inorganic semiconductorsexamples: Qdot 565, Qdot 605Fluorochromes流式细胞技术荧光抗体课件4Some Common FluorochromesFluorochromeType of moleculeFluorescein isothyocyanate (FITC)Small organicAlexa Fluor 488Small organicPhycoerythrin (PE)ProteinPE-CyTM5Protein tandemPeridinin chlorophyll protein (PerCP)ProteinPerCP-CyTM5.5Protein tandemPE-CyTM7Protein tandemAllophycocyanin (APC)ProteinAlexa Fluor 647Small organicAPC-CyTM7Protein tandemBDTM APC-H7Protein tandemBD HorizonTM V450Small organicPacific BlueTMSmall organicBD HorizonTM V500Small organicAmCyanProteinFluorochromes流式细胞技术荧光抗体课件5Small Organic FluorochromesAdvantages Low molecular weight Easy to conjugatedirect attachment to free amino groups on mAb Excellent stability Extremely consistent emission spectraDisadvantages Small Stokes Shift (50100 nm) Tend to be less brightFluorochromes流式细胞技术荧光抗体课件6Protein FluorochromesAdvantages Good stability Consistent emission spectra Medium Stokes Shift (75200 nm) Tend to be more brightDisadvantages High molecular weight More difficult to conjugateintermediaries needed to attach to mAbFluorochromes流式细胞技术荧光抗体课件7Tandem Dye FluorochromesAdvantages Very large Stokes Shift (150300 nm) Tend to be very brightoften brighter than the fluorescent protein donor Disadvantages High molecular weight (similar to fluorescent protein) Difficult to make consistently (lot-to-lot variation in emission properties) Harder to conjugate (same as fluorescent protein) Some tandems have poor stabilityFluorochromes流式细胞技术荧光抗体课件8Nanocrystal FluorochromesAdvantages Large Stokes Shift (100500 nm) Tend to be very bright Emission peaks are consistent and narrow, and do not change with variations in the excitation source Highly resistant to photobleaching Nanocrystals share biophysical and conjugation properties Disadvantages Difficult to conjugate Instability of bindings Cytotoxicity Wide excitation range produces cross-laser spilloverFluorochromes流式细胞技术荧光抗体课件9Excitation and Emission Excitation wavelengths determine lasers that can excite the fluorochrome. Emission wavelengths determine filters and PMTs that can measure the emission signal.Fluorochromes流式细胞技术荧光抗体课件10Know Your CytometerCytometer ConfigurationBD FACSCantoTM II 4-2-2 configuration is shown below BDTM LSR II 4-2-2 configuration is similar4 detectors for blue laser 2 detectors for red laser 2 detectors for violet laser Fluorochromes流式细胞技术荧光抗体课件11Typical Excitation and EmissionBD FACSCanto II 4-2-2 (BD LSR II 4-2-2 is similar)Detector Range Violet Laser 405 nmBlue Laser 488 nmRed Laser 633 nm410490 nmBD Horizon V450 Pacific BlueTM500560 nmBD Horizon V500 AmCyan515545 nmFITCAlexa Fluor 488564606 nmPE650670 nmAPCAlexa Fluor 647670735 nmPerCP-Cy5.5PE-Cy5PerCP750810 nmPE-Cy7BD APC-H7APC-Cy7Fluorochromes流式细胞技术荧光抗体课件12Fluorochrome Use Depends on the Cytometer Configuration 6-color 8-color More than 8 colorsFITC, Alexa Fluor 488FITC, Alexa Fluor 488FITC, Alexa Fluor 488PEPEPEPE-Texas Red, PE-Alexa Fluor 610, or PE-Alexa Fluor 594PE-Cy5, PerCP,or PerCP-Cy5.5PE-Cy5, PerCP,or PerCP-Cy5.5PE-Cy5, PerCP,or PerCP-Cy5.5PE-Cy7PE-Cy7PE-Cy7APC, Alexa Fluor 647APC, Alexa Fluor 647APC, Alexa Fluor 647APC-Cy5.5, Alexa Fluor 680, Alexa Fluor 700APC-H7, APC-Cy7APC-H7, APC-Cy7APC-H7, APC-Cy7BDHorizon V500, AmCyanBDHorizon V500, AmCyanBDHorizon V450, Pacific BlueBD Horizon V450, Pacific BluePacific Orange, Q-dotsFluorochromes流式细胞技术荧光抗体课件13Fluorochrome BrightnessThe brightness of a fluorochrome depends on two factors: Molar Extinction Coefficient ( ) measures how well a fluorochrome absorbs energy. Quantum Yield (Qy) is the ratio of photons emitted to photons absorbed.Brightness = x QyRelative Brightness =Brightness of PEBrightnessFluorochromes流式细胞技术荧光抗体课件14Some Fluorochromes are MUCH Brighter PE is 50 x brighter than FITC and 10 x brighter than APC. APC is 5x brighter than Pacific Blue. Extinction coefficient is more significant than quantum yield in determining brightness. FluorochromeMolar Extinction Coefficient (mol-1 x cm-1)Quantum YieldRelative Brightness (to PE)PE0.98100.00%PE-Cy50.881.63%PerCP320000NA16.66%APC2320000.688.21%FITC670000.50 1.74%Pacific Blue360000.801.5%Fluorochromes流式细胞技术荧光抗体课件15DD = difference between the medians of the positive and negative populationsW = spread (2 x rSD) of the negative populationStain IndexStain Index =Stain index is a practical way to characterize the brightness of a marker with respect to a given optical configuration.DWWStain index can also be used to characterize the sensitivity of a fluoresence parameter.Fluorochromes流式细胞技术荧光抗体课件16Typical CD4 Stain IndexesBD LSR II FluorochromeCD4 CloneFilterSetStain IndexRelative BrightnessPERPA-T4585/40305100.00%PE-Cy5RPA-T4695/4019881.63%PerCPRPA-T4695/403016.66%APCRPA-T4660/202638.21%FITCRPA-T4530/30431.74%Pacific BlueRPA-T4440/40631.5%APC has a higher CD4 stain index than PE-Cy5, but approximately 1/10th the relative brightness.Fluorochromes流式细胞技术荧光抗体课件17Typical CD4 Stain IndexesBD FACSCanto II FluorochromeCD4 CloneFilterSetStain IndexRelative BrightnessPESK3585/42467100.00%PE-Cy581.63%PerCPSK3695/405916.66%APCSK3660/203848.21%FITCSK3530/30581.74%Pacific BlueSK3450/50131.5%FITC has approximately the same CD4 stain index as PerCP, but approximately 1/10th the relative brightness.Fluorochromes流式细胞技术荧光抗体课件18Stain Index Factors Stain index is dependent on the optical configuration and additional performance factors. Factors that can affect stain index include:Laser wavelength and powerDetector rangeDetector efficiencyBackground signal Dont depend on published valuesmeasure stain index on your own system.Fluorochromes流式细胞技术荧光抗体课件19CD4 Stain Indexes Across CytometersStain Index ExerciseFluorochromes流式细胞技术荧光抗体课件20Fluorescence SpilloverEmission of FITC in PE channelFluorochromes流式细胞技术荧光抗体课件21Significant Spillovers on 4-2-2 ConfigurationFluorochromes流式细胞技术荧光抗体课件22Spillover Decreases SensitivityWithout CD45 AmCyanWith CD45 AmCyanCD19 FITCSpillover can significantly increase the variability of negative and dim populations, even after compensation is applied.Fluorochromes流式细胞技术荧光抗体课件23Lost Population due to SpilloverLymphocytes stained with CD45 FITC and CD4 PECD45 FITC causes dim CD4+CD45+ to be difficult to distinguish due to significant FITC spillover into PE.Lymphocytes stained with CD45 PerCP and CD4 PECD45 PerCP allows dim CD4+CD45+to be distinguished from backgrounddue to minimal PerCP spillover into PE.Fluorochromes流式细胞技术荧光抗体课件24Tandem Dye IssuesUse tandem dyes in research applications with consideration of their technical limitations. APC-Cy7 (and to a lesser extent PE-Cy7) can degrade in the presence of light, fixation, and elevated temperature. Degradation causes lower emission in the Cy7 detector and higher emission in the detector of the parent dye (APC or PE). APC-H7, APC-Cy7, and PE-Cy7 performance can be affected by polystyrene tubes.Fluorochromes流式细胞技术荧光抗体课件25Tandem DegradationFalse PositivesFalse positives in APC channel reduced in absence of APC-Cy7With CD8 APC-Cy7WithoutCD8 APC-Cy7Fluorochromes流式细胞技术荧光抗体课件26Tandem Degradation Over Time0 hours2 hours20 hoursPECD3 PE-Cy5CD8 PE-Cy7Time Sample Left in LightPEPEFluorochromes流式细胞技术荧光抗体课件27APC-H7 Is More Stable Than APC-Cy7 Comparison of Sample Stability(in BD Stabilizing Fixative at RT)0501001502002500124682448Hours of light exposure% SpilloverCD4 APC-H7CD8 APC-H7CD4 APC-Cy7CD8 APC-Cy7Fluorochromes流式细胞技术荧光抗体课件28Tandem Dye Recommendations When using APC-Cy7 and PE-Cy7, beware of fixative and light instability issues. If problems arise when using polystyrene with APC-H7, APC-Cy7, or PE-Cy7, try switching to K-resin or polycarbonate. PerCP-Cy5.5 is less susceptible to instability than APC-Cy7 and PE-Cy7. BD offers APC-H7 conjugated antibodies that are more stable than APC-Cy7 conjugates and have less spillover into the APC detector.Fluorochromes流式细胞技术荧光抗体课件29New Violet Laser FluorochromesBD Horizon V450 Maximum excitation at 404 nm, emission peak of 448 nm. BD Horizon V450 reagents exhibit performance comparable to Pacific Blue reagents as measured by Stain Index. Improved stability with fixation compared to Pacific Blue.Fluorochromes流式细胞技术荧光抗体课件30New Violet Laser FluorochromesBD Horizon V500 Maximum excitation at 415 nm, emission peak of 500 nm. Provides significantly reduced spillover into the FITC channel compared to AmCyanV500 has low excitation with the blue laser. Improved stability with fixation compared to AmCyan.Fluorochromes流式细胞技术荧光抗体课件31BD Horizon V500 Low Spillover into FITCV500 stained cellsAmCyan stained cellsData is uncompensated.Fluorochromes流式细胞技术荧光抗体课件32Factors Affecting Reagent Performance Relative brightness of the fluorochrome Number of fluorochromes per antibody Expression levels on (or in) the cells of interest Background contributions Spillover Photostability Reagent environment Cytometer configurationFluorochromes流式细胞技术荧光抗体课件33Non-Conjugated Fluorescent Dyes These dyes bind directly to cell components. Viability dyes discriminate live cells from dead cells. examples: 7-AAD, TO-PRO-3, PI, DAPI, and the LIVE/DEAD series. Proliferation dyes are used to study cell division. examples: CSFE, PKH 67, and BD Horizon VPD 450. DNA dyes are used for DNA cell-cycle anaylsis. examples: PI, DAPI, and Hoechst 33342. Reporter dyes are used to study gene expression. examples: mCherry and GFP.Fluorochromes流式细胞技术荧光抗体课件34Non-Conjugated Fluorescence Dye DetectionBD FACSCanto II 4-2-2 and BD LSR II 4-2-2Detector Range Violet Laser 405 nmBlue Laser 488 nmRed Laser 635 nm410490 nmHoechst 33342, DAPI,Horizon VPD 450, LIVE/DEAD Violet500560 nmLIVE/DEAD Aqua515545 nmCSFE, GFP,LIVE/DEAD Green564606 nmPI650670 nmTO-PRO-3670735 nm7-AAD750810 nmLIVE/DEAD Near-IRmCherry is best detected with a 561-nm laser option and a 590630 detector range.Fluorochromes流式细胞技术荧光抗体课件35BD Fluorescence Spectrum V Use the Fluorescence Spectrum Viewer to help choose fluorochromes based on: the number of colors you need in your experiment your cytometer configuration spillover issues